Disease in Plants
Plants are hosts to any of various infectious diseases (numbering in the thousands) caused by a vast array of respective phytopathogenic fungi, bacteria, viruses, and nematodes, for example, these pathogens are responsible for significant crop losses worldwide, resulting from both infection of growing plants and destruction of harvested crops. The most widely practiced methods of reducing damage caused by such pathogens involve the use of various chemical agents that kill or attenuate the action of the respective pathogen. Unfortunately, many plant pathogens develop resistance to such chemicals, and some plant pathogens (especially viruses) are not susceptible to control by chemical means. In addition, many of the chemical agents used are broad-spectrum toxins, and may cause serious environmental damage, as well as toxicity in humans and animals.
Plant breeding and, more recently, genetic engineering techniques also have been employed to combat plant pathogens. In certain instances, breeders and molecular biologists have successfully engineered resistance in plants to certain pathogens. In the past few years, a number of plant R (resistance) genes have been isolated from plants. When introduced into otherwise susceptible crops, these R genes produce enhanced resistance to certain pathogens. For example, U.S. Pat. No. 5,571,706 describes the isolation of the tobacco N gene that confers enhanced resistance to Tobacco Mosaic Virus. However, whereas conventional breeding and genetic engineering approaches reported to date can successfully enhance pathogen resistance in plants, the approaches typically address problems caused by only one target pathogen, or a small number of closely related pathogens. As a result, while crops produced using these approaches may have enhanced protection against the target pathogen, conventional chemical agents still must be used to control other pathogens.
Antimicrobial Peptides
In the past two decades a large number of natural polypeptides (“peptides”) with a broad range of antimicrobial activities have been discovered (for reviews see Hancock and Lehrer, Trends Biotechnol. 16:22–28, 1998; Hancock, Mol. Microbiol. 12:951–958, 1994; and Nicholas and Mor, Ann. Rev. Microbiol. 49:277–304, 1995). The endogenous antimicrobial peptides of plants and animals typically consist of 12–45 amino acids, and are amphipathic molecules having a net positive charge (cationic) at physiological pH. Although cationic antimicrobial peptides (CAPs) are structurally diverse, they fall into two general classes of structures: α-helical peptides, such as the cecropins and magainans, and β-sheet peptides stabilized by intramolecular disulphide bonds, such as the defensins, protegrins, and tachyplesins. Hancock and Lehrer, Trends Biotechnol. 16: 22–28, 1998; Zasloff, Curr. Opin. Immunol. 4:3–7, 1992; Cociancich et al., Biochem. J. 300:567–575 1994; and Piers and Hancock, Mol. Microbiol. 12:951–958, 1994. Natural CAPs vary greatly in their respective spectra of biological activities, including killing bacteria (Gram-positive and -negative), fungi, protozoa, and even viruses. CAPs normally kill susceptible microorganisms in vitro at concentrations from 0.25 μg/mL to 4 μg/mL (Hancock and Lehrer, Trends Biotechnol. 16: 22–28, 1998), providing exciting possibilities in the face of the declining efficiency of conventional antibiotics. Furthermore, the expression of CAP in plants may introduce broad-spectrum resistance to phytopathogenic microorganisms. Jaynes, Plant Science 89:43–53, 1993; and Misra and Zhang, Plant Physiol. 106: 977–981, 1994.
Insect cecropins represent a family of small, highly basic, α-helical antimicrobial peptides that form an important component in the immune response of insects. Bohman and Hultmark, Annu. Rev. Microbiol. 41:103–126, 1987. Cecropins isolated from the giant silk moth, Hyalophora cecropia, contain about 35 amino acid residues with amphipathic N-termini and hydrophobic C-termini (van Hofsten et al., Proc. Natl. Acad. Sci. USA 82:2240–2243, 1985). All cecropins are potent antibacterials in vitro, and several members of this family are particularly powerful in vitro against a number of plant pathogenic bacteria. Hultmark et al., Eur. J. Biochem. 127:207–217, 1982; Jaynes et al., BioEssays 6:263–270, 1987; and Nordeen et al., Plant Sci. 82:101–107, 1992.
Another antibacterial peptide, mellitin, containing 26 amino acids, is the major component of bee venom. As opposed to cecropins, mellitin has a predominantly hydrophobic N-terminus with an amphipathic C-terminus. Habermann, Science 177:314–322, 1972. Although mellitin possesses potent antimicrobial activity, its powerful hemolytic activity (Tosteson et al., J. Membr. Biol. 87:35–44, 1985) makes it unsuitable for therapeutic use and likely a poor candidate for transgenics.
In view of the above, there is a need for plants having enhanced resistance to a wider than normal spectrum of pathogens, including bacterial and fungal pathogens.